Discovery of substituted N-phenyl nicotinamides as potent inducers of apoptosis using a cell- and caspase-based high throughput screening assay

Article abstract of DOI:10.1021/jm0205200

By applying a novel cell- and caspase-based HTS assay, a series of N-phenyl nicotinamides has been identified as a new class of potent inducers of apoptosis. Through SAR studies, a 20-fold increase in potency was achieved from a screening hit N-(4-methoxy-2-nitrophenyl)-pyridine-3-carboxamide (1) to lead compound 6-methyl-N-(4-ethoxy-2-nitrophenyl)pyridine-3- carboxamide (10), with an EC₅₀ of 0.082 μM in the caspase activation assay in T47D breast cancer cells. The N-phenyl nicotinamides also were found to be active in the growth inhibition assay where compound 10 had a GI₅₀ value of 0.21 μM in T47D cells. More importantly, compound 10 was found to be equipotent in MES-SA cells and paclitaxel-resistant, p-glycoprotein overexpressed MES-SA/DX5 cells. Compounds 1 and 6-chloro-N-(4-ethoxy-2-nitrophenyl)pyridine-3-carboxamide (8), a more potent analogue, were found to arrest T47D cells in G₂/M phase of the cell cycle followed by induction of apoptosis as measured by flow cytometry. Compound 8, which was more potent than 1 in the caspase activation assay, also was found to be more potent in G₂/M arrest and apoptosis assay. These data confirm that the cell-based caspase activation assay is useful for screening for inducers of apoptosis, as well as for SAR studies and lead optimization. Upon further characterization, N-phenyl nicotinamides were found to be inhibitors of microtubule polymerization in vitro. The identification of N-phenyl nicotinamides as a novel series of inducers of apoptosis demonstrates that our cell- and caspase-based HTS assay is useful for the discovery and optimization of potentially novel anticancer agents.

Full text of DOI:10.1021/jm0205200

2474
J . Med. Chem. 2003, 46, 2474-2481
Discover y of Su bstitu ted N-P h en yl Nicotin a m id es a s P oten t In d u cer s of
Ap op tosis Usin g a Cell- a n d Ca sp a se-Ba sed High Th r ou gh p u t Scr een in g Assa y
Sui Xiong Cai,* Bao Nguyen, Shaojuan J ia, J ohn Herich, J ohn Guastella, Sanjeeva Reddy, Ben Tseng,
J ohn Drewe, and Shailaja Kasibhatla
Cytovia, Inc., a Subsidiary of Maxim Pharmaceuticals, Inc., 6650 Nancy Ridge Drive, San Diego, California 92121
Received November 15, 2002
By applying a novel cell- and caspase-based HTS assay, a series of N-phenyl nicotinamides
has been identified as a new class of potent inducers of apoptosis. Through SAR studies, a
20-fold increase in potency was achieved from a screening hit N-(4-methoxy-2-nitrophenyl)-
pyridine-3-carboxamide (1) to lead compound 6-methyl-N-(4-ethoxy-2-nitrophenyl)pyridine-3-
carboxamide (10), with an EC50 of 0.082 µM in the caspase activation assay in T47D breast
cancer cells. The N-phenyl nicotinamides also were found to be active in the growth inhibition
assay where compound 10 had a GI50 value of 0.21 µM in T47D cells. More importantly,
compound 10 was found to be equipotent in MES-SA cells and paclitaxel-resistant, p-
glycoprotein overexpressed MES-SA/DX5 cells. Compounds 1 and 6-chloro-N-(4-ethoxy-2-
nitrophenyl)pyridine-3-carboxamide (8), a more potent analogue, were found to arrest T47D
cells in G2/M phase of the cell cycle followed by induction of apoptosis as measured by flow
cytometry. Compound 8, which was more potent than 1 in the caspase activation assay, also
was found to be more potent in G2/M arrest and apoptosis assay. These data confirm that the
cell-based caspase activation assay is useful for screening for inducers of apoptosis, as well as
for SAR studies and lead optimization. Upon further characterization, N-phenyl nicotinamides
were found to be inhibitors of microtubule polymerization in vitro. The identification of N-phenyl
nicotinamides as a novel series of inducers of apoptosis demonstrates that our cell- and caspase-
based HTS assay is useful for the discovery and optimization of potentially novel anticancer
agents.
In tr od u ction
based high throughput screening (HTS) system for
inducers of apoptosis8 using our novel fluorescent
caspase substrates.9,10 The screening uses a mono-
DEVD-tetrapeptide rhodamine 110-based caspase-3
substrate N-(Ac-DEVD)-N′-ethoxycarbonyl-R110, which
is a profluorescent molecule that exhibits little back-
ground signal before cleavage.10 The substrate, after
cleavage by caspases, fluoresces at a long wavelength
(525 nm), thereby avoiding interference from cellular
fluorescence and from the approximately 1% of test
compounds found to fluoresce at short wavelengths
(<500 nm, data not shown). The screening assay is
extremely flexible: it can be applied to any cell type or
cell line that expresses caspases and can be adapted to
screen for either apoptosis inducers or inhibitors. Fur-
thermore, the assay can detect any apoptosis inducer
as long as it acts by a caspase-mediated mechanism and
triggers apoptosis at a point upstream of caspase-3
activation. In addition, since a compound active in this
assay must interact either with cell surface or intrac-
ellular targets in order to induce apoptosis and activate
caspases, the assay also avoids the false positives
observed with some enzyme screening assays due to
precipitation or crystallization of screening compounds
on the enzymes as reported recently by McGovern et
al.11 Herein we report the discovery, characterization,
and preliminary SAR of substituted N-phenyl nicotina-
mides as potent inducers of apoptosis using our cell- and
caspase-based apoptosis HTS assay.
Apoptosis or programmed cell death is a normal
process that organisms use to eliminate unwanted and
excessive cells and is important in animal development,
as well as in tissue homeostasis.1 However, improperly
regulated apoptosis can lead to many pathological
conditions. Inadequate or abnormal inhibition of apo-
ptosis, which leads to unchecked cell proliferation,
results in cell accumulation and is a hallmark of cancer.2
Excessive apoptosis, on the other hand, could lead to
organ failure and neurodegenerative diseases.3 Apop-
tosis can be initiated by many agents, including TNF-R
and FAS ligand, as well as by chemotherapy and
radiation. The mechanism of apoptosis involves a cas-
cade of initiator and effector caspases that are activated
sequentially. Caspases are a family of cysteine proteases
that require aspartic acid residues at the P1 position of
substrates for cleavage.4 Among these caspases, caspase-
3, -6, and -7 are key effector caspases that cleave
multiple protein substrates in cells, leading irreversibly
to cell death.5
Since many of the current anticancer agents are
known to kill tumor cells through the induction of
apoptosis,6,7 we are interested in the discovery and
development of inducers of apoptosis as potential anti-
cancer agents. Toward this goal, we developed a cell-
* Maxim Pharmaceuticals, Inc. 6650 Nancy Ridge Drive, San Diego,
CA., 92121. Phone: 858-202-4006. Fax: 858-202-4000. E-mail: scai@
maxim.com.
10.1021/jm0205200 CCC: $25.00 © 2003 American Chemical Society
Published on Web 04/29/2003

N-Phenyl Nicotinamides as Inducers of Apoptosis
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 12 2475
Sch em e 1
Sch em e 2
Ch a r t 1
Resu lts a n d Discu ssion
F igu r e 1. Fluorescent micrographs of J urkat cells treated
with compound 1 and stained with a fluorescent DNA probe,
Syto16. (A) Control cells. (B) Cells treated with 5 µM of
compound 1 for 24 h, showing shrunken and fragmented
nuclei, as well as condensed chromatin spindles.
Ch em istr y. N-(4-Methoxy-2-nitrophenyl)pyridine-3-
carboxamide 1 was obtained originally from a com-
mercial compound library. It was prepared by reaction
of nicotinoyl chloride with 4-methoxy-2-nitroaniline and
was isolated as an orange solid. Compounds 3, 4, 5, 8,
and 9 were prepared similar to 1 from the corresponding
substituted nicotinoyl chlorides or acyl chloride and
anilines (Scheme 1). Compound 7 and 10 were prepared
from the corresponding acids and anilines in the pres-
ence of cyanuric chloride (Scheme 2). Compounds 2 and
6 were obtained from commercial sources, and their
structures were confirmed by 1H NMR spectroscopy
(Chart 1).
induce apoptosis and activate caspase in T47D cells with
a ratio of 6 over untreated cells, and an EC50 value of
1.6 µM.
Ch a r a cter iza tion of Com p ou n d 1. The ability of
compound 1 to induce apoptosis was confirmed in a
nuclear fragmentation assay. J urkat cells were treated
with 5 µM of compound 1 for 24 h followed by staining
with Syto16, a DNA stain which allows the visualiza-
tion of nuclear morphology. The compound-treated cells
were characterized by shrunken and fragmented nuclei
with condensed chromatin (Figure 1B), which is a
hallmark of caspase-mediated apoptosis, as well as by
condensed mitotic spindles crucial to the transition to
metaphase, similar to the effect of vinca alkaloids.12 In
contrast, the nuclei of J urkat cells treated with vehicle
control (DMSO) appeared to be normal with dispersed
chromatin that is moderately stained with Syto16
(Figure 1A).
The apoptosis-inducing activities of compound 1 were
also characterized by treatment of T47D cells with 10
µM of compound 1 for 48 h, staining with propidium
iodide, and analysis by flow cytometry. An increase in
the G2/M DNA content (M4) in cells treated with
compound 1 was observed, as shown in Figure 2B. The
sub-diploid DNA content (M1, apoptotic sub-G1 area) of
cells increased from 2.6% to 25.1% upon compound
treatment, indicating the presence of apoptotic cells
which have undergone DNA degradation and nuclear
fragmentation. The results showed that treatment of
T47D cells by compound 1 for 48 h results in cell cycle
arrest in the G2/M phase, as well as induction of
apoptosis.
HTS Assa ys. N-(4-Methoxy-2-nitrophenyl)pyridine-
3-carboxamide 1 was identified as an inducer of apo-
ptosis from a commercial compound library using our
cell-based apoptosis induction HTS assay in T47D
breast cancer cells. Briefly, human breast cancer T47D
cells, in a 96-well microtiter plate containing 10 µM of
test compound, were incubated for 24 h for the induction
of apoptosis. Caspase-3 fluorogenic substrate N-(Ac-
DEVD)-N′-ethoxycarbonyl-R11010 was then added to
cells, and the samples were mixed by agitation and
incubated at room temperature for 3 h. Using a fluo-
rescent plate reader, employing excitation at 485 nm
and emission at 525 nm, the fluorescence was measured,
and the amount of caspase activation was determined.
Compounds that induce apoptosis and activate the
caspases yield a fluorescent signal higher than the
background (signal/background ratio). Compounds found
to give a ratio of >3 are considered active and retested
in triplicate for confirmation. Compounds confirmed to
be active are then tested at several concentrations to
provide a dose response and the caspase activation
activity (EC50) calculated. Compound 1 was found to

2476 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 12
Cai et al.
Ta ble 1. Caspase Activation Activity of Substituted N-Phenyl Nicotinamides and Analogues
EC50 (µM)a
ZR75-1
A
B
D
R1
R2
R3
R4
T47D
DLD-1
1
2
3
4
5
6
7
8
C
C
C
C
C
C
N
C
C
C
NA
NA
N
N
N
N
CH
N
CH
N
CH
N
NA
NA
C
C
C
C
N
C
C
C
C
C
NA
NA
H
H
Cl
H
H
H
NA
H
H
H
H
H
H
Cl
NA
H
H
Br
H
H
H
H
H
H
H
Me
Me
Me
Me
Me
Et
Et
Et
Et
Et
1.6 ( 0.1
>10
0.88 ( 0.02
5.4 ( 0.1
>10
2.9 ( 0.1
>10
>10
>10
0.69 ( 0.03
>10
0.20 ( 0.01
>10
0.24 ( 0.02
5.7 ( 0.3
0.082 ( 0.005
0.17 ( 0.01
0.014 ( 0.001
0.30 ( 0.01
>10
0.69 ( 0.20
>10
0.11 ( 0.02
>10
0.11 ( 0.01
3.0 ( 0.3
0.052 ( 0.006
0.13 ( 0.01
0.006 ( 0.001
0.19 ( 0.02
>10
H
Cl
Cl
Me
NA
NA
0.38 ( 0.01
>10
9
10
11
12
H
NA
NA
0.11 ( 0.01
0.15 ( 0.01
0.19 ( 0.01
NA
NA
NA
NA
a
Data are the mean of three or more experiments and are reported as mean ( standard error of the mean (SEM). NA ) not applied.
microtiter plate containing various concentration of test
compound and incubated at 37 °C for 24 h. After
incubation, the samples were treated with the N-(Ac-
DEVD)-N′-ethoxycarbonyl-R110 fluorogenic substrate.
The caspase activation activity (EC50) of compound 1
and its analogues in the three cancer cell lines is
summarized in Table 1.
Table 1 shows that compound 1 has an EC50 for
caspase activation of 1.6, 0.88, and 2.9 µM in T47D,
ZR75-1 and DLD-1 cells, respectively. Substitution by
a bromine in the 5-position (2) or a chlorine in the
2-position (3) of pyridine ring resulted in reduction of
potency by >6-fold in the T47D cells, suggesting a space
limited pocket around the 2 and 5-position. Substitution
at the 6-position of the pyridine ring by a chlorine (4)
increased the potency by >2-fold, suggesting the pres-
ence of a binding pocket in that position. Interestingly,
the 4-pyridyl analogue (5) was found to be at least 6
times less active than 1, suggesting that the position of
nitrogen in the 3-pyridyl group of 1 plays an important
role for interaction with its target, probably by hydrogen
bonding. Replacing the methoxy group in the 4-position
of the phenyl ring by an ethoxy group (6) resulted in a
8-fold increase in potency, suggesting that there is a
hydrophobic binding pocket in that position. The 2-py-
ridyl analogue (7) was >50 times less active than 6,
again suggesting the importance of the position of
nitrogen in the pyridyl group. A combination of 6-chlo-
F igu r e 2. Drug-induced cell cycle arrest and apoptosis in
rine in the pyridine ring with 4-ethoxy group in the
T47D cells as measured by flow cytometric analysis. The x-axis
phenyl ring yielded compound 8, which was >6-fold
is the fluorescence intensity and the y-axis is the number of
more potent than 1. The corresponding phenyl analogue
(9) was >20 times less active than 8, confirming the
importance of the nitrogen in the 3-pyridyl group.
Finally, replacement of the 6-chloro group in 8 by a
6-methyl group yielded compound 10, which was ap-
proximately 20-fold more potent than 1, the original
screening hit. Compared with reference compounds,
compound 10 is approximately 2-fold more potent in
caspase activation assays than T67 (11), a pentafluo-
rophenyl sulfonamide compound developed by Tularik
and currently in Phase II clinical trials,13 and 6-fold less
cells with that fluorescence intensity. (A) Control cells showing
most of the cells in G1 (M2). (B) Cells treated with 10 µM of
compound 1 for 48 h showing a reduction in the G1 (M2), an
increase in the G2/M (M4) and sub-diploid DNA content of cells
(M1).
Str u ctu r e Activity Rela tion sh ip (SAR) Stu d ies.
The cell-based caspase HTS assay was used to test
analogues of compound 1 for SAR studies. A panel of
three different cell lines was used for these experiments.
Briefly, human breast cancer T47D, ZR75-1, and a
colorectal DLD-1 cell line were placed in a 384-well

N-Phenyl Nicotinamides as Inducers of Apoptosis
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 12 2477
Ta ble 2. Comparison of Caspase Activation Activity and Inhibition of Cell Proliferation Activity of Substituted N-Phenyl
Nicotinamides
T47D
DLD-1
a
b
a
b
EC50 (µM)
GI50 (µM)
GI50/EC50
EC50 (µM)
GI50 (µM)
GI50/EC50
1
6
10
1.6 ( 0.1
0.20 ( 0.01
0.082 ( 0.005
7.0 ( 0.5
0.56 ( 0.05
0.21 ( 0.03
4.4
2.8
2.6
2.9 ( 0.1
0.19 ( 0.02
0.11 ( 0.01
10.0 ( 0.1
0.90 ( 0.02
0.53 ( 0.05
3.4
4.7
4.8
a
b
From Table 1. Data are the mean of three experiments and are reported as mean ( standard error of the mean (SEM).
Ta ble 3. GI50 Assay of Compound 10 in Comparison with
Paclitaxel in MES-SA and MES-SA/DX5 Cells
Ch a r t 2
GI50 (µM)a
compound
MES-SA
MES-SA/DX5
0.074 ( 0.008
>10
10
0.073 ( 0.008
0.063 ( 0.012
paclitaxel
a
Data are the mean of three or more experiments and are
reported as mean ( standard error of the mean (SEM).
taxanes, has made microtubules the single best cancer
drug target identified to date.14 However, one of the
most serious problems of currently used anticancer
agents is multidrug resistance. Many tumor cells over-
express the p-glycoprotein, a plasma membrane trans-
porter with a broad substrate specificity that includes
many of the anticancer agents.15 This transporter ef-
ficiently removes the anticancer agents from the interior
of the cell, thus reducing or eliminating the effects of
these agents. Therefore, it is important to discover and
develop novel anticancer agents that are active against
multidrug resistant cancers. Using the cell proliferation
assay, compound 10 was tested against both the human
uterus sarcoma MES-SA cell and its doxorubicin-
resistant variant, MES-SA/DX5. As shown in Table 3,
compound 10 had similar GI50 values in MES-SA (0.073
µM) and MES-SA/DX5 (0.074 µM) cells. Therefore, the
growth inhibition activity of compound 10 is not affected
by the overexpression of mdr1 p-glycoprotein in the
MES-SA/DX5 cells. In comparison, the activity of pa-
clitaxel in the MES-SA/DX5 was reduced by >160-fold.
These data suggest that compound 10 should be effec-
tive in tumors that are resistant to paclitaxel due to
overexpression of mdr1.
The apoptosis-inducing activity of compound 8 was
also tested by flow cytometry similar to that of com-
pound 1. T47D cells treated with 1 µM of compound 8
for 48 h also showed an increase in G2/M DNA content
(M4) (Figure 3B). The sub-diploid DNA content of cells
increased from 3.3% to 56.1% with compound treatment,
indicating that >50% of the cells were apoptotic. In
comparison, compound 1 at 10 µM increased the sub-
diploid DNA content of cells from 2.6% to 25.1%. These
results show that compound 8 is about 10-fold more
potent than compound 1 as an inducer of apoptosis as
measured by the flow cytometry, similar to the results
obtained in the caspase activation assay, confirming
that the cell-based caspase activation assay is useful for
SAR studies.
potent than the well-known antimitotic agent, colchicine
(12) (Chart 2).
The activities of these compounds toward the breast
cancer cell line ZR75-1 and colon cancer cell line DLD-1
was roughly the same as their activity toward T47D
cells. ZR75-1 cells were slightly more sensitive (about
2-fold more as measured by the EC50 value) to the
compounds than T47D cells in this assay. DLD-1 cells
were slightly less sensitive (about 1.5-fold less as
measured by the EC50 value) to the compounds than
T47D cells.
Selected compounds were also tested by a more
traditional inhibition of cell proliferation (GI50) assay
to confirm that the active compounds can inhibit tumor
cell growth, as well as to determine whether there is a
correlation between the activity from the caspase acti-
vation assay and the cell proliferation assay. Briefly,
T47D and DLD-1 cells in a 96-well microtiter plate were
treated with serial concentrations of test compound. The
survival profiles of the cells were quantitated using the
CellTiter-Glo (Promega, Madison, WI) according to the
manufacturer’s protocol. This assay is a homogeneous
method of determining the number of viable cells in
culture based on the quantitation of ATP, which rep-
resents the presence of metabolically active cells. GI50
values were calculated from dose-response curves using
XLFit3 (IDBS) software. The GI50 (µM), along with the
EC50 data and the ratio of GI50/EC50, are summarized
in Table 2.
Table 2 shows that compounds 1, 6, and 10 all
inhibited the growth of tumor cells. Compound 10 has
a GI50 value of 0.21 µM and 0.53 µM in T47D and DLD-1
cells, respectively. The compound that is more active
in the apoptosis induction assay, as measured by
caspase activation, also is more potent in the growth
inhibition assay. The ratio of GI50/EC50 for the three
compounds in T47D and DLD-1 cells has a value of
around 3.5, suggesting a correlation between the caspase
activation activity and inhibition of cell proliferation
activity for these compounds. Overall, these data indi-
cate that the cell-based caspase activation HTS assay
not only is useful for the identification of apoptosis
inducers, but also can be used for subsquent optimiza-
tion and SAR studies of screening hits.
Since tubulin inhibitors are known to arrest cells in
the G2/M phase, we hypothesized that compound 1 and
its analogues might interact with tubulin. In addition,
we also observed some structural similarity between
compound 1 and the known tubulin inhibitor sulfona-
mide 11,16 which was reported to covalently modify
â-tubulin due to the chemically reactive pentafluorophe-
nylsulfonyl group. Therefore, we tested the ability of
The success of microtubule interacting antimitotic
agents in the clinic, such as the vinca alkaloids and the

2478 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 12
Cai et al.
cally reactive group. For instance, there is no potentially
reactive group in the phenyl group and the pyridyl group
in compound 1. The chloro group in compound 8 might
be considered potentially reactive. However, replace-
ment of the chloro group by a methyl group resulted in
10, which was 3-fold more active than 8, indicating that
the activity is not dependent on potential reactivity of
the chloro group. Since tubulin is an important compo-
nent of every cell, a tubulin inhibitor that does not
covalently modify and bind to tubulin might be expected
to have fewer side effects and toxicity.
Con clu sion s
In conclusion, using a cell- and caspase-based HTS
assay, a series of substituted N-phenyl nicotinamides
has been identified as potent inducers of apoptosis. SAR
studies of N-phenyl nicotinamides indicate that the
3-pyridyl group is very important for its activity and
substitution in the 6-position by a chloro or methyl
group resulted in increased potency. Substitution at the
4-position of the phenyl group also is important, and
changing the methoxy group to an ethoxy group resulted
in increased potency. Through the SAR studies, a 20-
fold increase in potency was obtained from compound
1 to compound 10. The N-phenyl nicotinamides were
also found to be active in the traditional growth inhibi-
tion assays. More importantly, compound 10, the most
potent compound in this series, was found to be active
in paclitaxel-resistant, p-glycoprotein overexpressed
MES-SA/DX5 cells.
N-Phenyl nicotinamides were found to arrest cells in
G2/M and induce apoptosis. Compound 8, which is more
potent than 1 in the caspase activation assay, also was
found to be more potent as an inducer of apoptosis in
the flow cytometric assay. These data, together with the
growth inhibition data, confirm that the cell-based
caspase activation assay is useful for screening for
inducers of apoptosis, as well as for SAR studies and
lead optimization. The N-phenyl nicotinamides were
found to be inhibitors of microtubule polymerization
similar to vinblastine. The identification of N-phenyl
nicotinamides as a new class of inducers of apoptosis
demonstrates that our cell- and caspase-based HTS
assay is useful for the discovery of potentially novel
anticancer agents. Since the N-phenyl nicotinamides are
active in paclitaxel-resistant cells and are not chemically
reactive, they might offer advantages over tubulin-
interacting drugs currently used in the clinic. Com-
pounds 8 and 10 are interesting leads for additional lead
expansion and in vivo studies and the results will be
reported in due course.
F igu r e 3. Drug-induced cell cycle arrest and apoptosis in
T47D cells as measured by flow cytometric analysis. The x-axis
is the fluorescence intensity and the y-axis is the number of
cells with that fluorescence intensity. (A) Control cells showing
most of the cells in G1 (M2). (B) Cells treated with 1 µM of
compound 8 for 48 h showing a reduction in the G1 (M2), an
increase in the G2/M (M4) and sub-diploid DNA content of cells
(M1).
compound 8 as an inhibitor of tubulin polymerization
in a tubulin polymerization assay. Compound 8 was
found to be a potent inhibitor of tubulin polymerization
with an IC50 value of 0.5 µM. In comparison, vinblastine,
a well-known tubulin polymerization inhibitor, was
found to have an IC50 value of 0.3 µM. Therefore,
compound 8 is about as potent as vinblastine for
inhibition of tubulin polymerization. Compound 8, and
other compounds in this series, most probably induces
apoptosis through the inhibition of tubulin polymeriza-
tion similar to that of vinblastine and pentafluorophenyl
sulfonamide 11.16
Although the N-phenyl nicotinamides and the sul-
fonamide 11 series of compounds are both tubulin
inhibitors and share some similar structural features,
suggesting that they might bind to tubulin in a similar
manner or at the same site, the SAR of N-phenyl
nicotinamides and sulfonamide 11 are very different.
The pentafluorophenyl group is essential for the activity
of 11, and it has been reported that the thiol group in
Cys-239 of â-tubulin can displace the p-F in the pen-
tafluorophenyl group, resulting in the covalent attach-
ment of 11 to â-tubulin.16 The importance of the
pentafluorophenyl group was confirmed from SAR stud-
ies of sulfonamide 11, and it has been reported that
replacement of the p-F in 11 by a chlorine or other group
eliminates activity.17 In comparison, the activity of
N-phenyl nicotinamides is not dependent on a chemi-
Exp er im en ta l Section s
Gen er a l Meth od s a n d Ma ter ia ls. The 1H NMR spectra
were recorded at 300 MHz. Chemical shifts are reported in
ppm (δ) and J coupling constants are reported in hertz.
Elemental analyses were performed by Numega Resonance
Labs, Inc. (San Diego, CA). Substituted anilines, nicotinic
acids, nicotinoyl chloride, and acyl chloride were obtained from
Aldrich or Lancaster and used as received. Reagent grade
solvents were used without further purification unless other-
wise specified. N-(Ac-DEVD)-N′-ethoxycarbonyl-R110 was pre-
pared as described in US patent 6,335,429.10 Colchicine (12)
was obtained from Sigma-Aldrich (St. Louis, MO). Sulfonamide
11 was prepared from pentafluorobenzenesulfonyl chloride and
3-fluoro-4-methoxyaniline as reported.17 Syto16 was obtained

N-Phenyl Nicotinamides as Inducers of Apoptosis
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 12 2479
from Molecular Probes (Eugene, OR). Human leukemia cancer
cells (J urkat cells), human breast cancer cells T47D and ZR75-
1, human colon cancer cells DLD-1, human uterus sarcoma
MES-SA cell, and human uterus sarcoma doxorubicin-resistant
MES-SA/DX5 cells were obtained from American Type Culture
Collection (Manasas, VA). Tubulin was obtained from Cytosk-
eleton (Boulder, CO).
for preparation of compound 10 (52%). 1H NMR (CDCl3): 12.54
(s, 1H), 8.92 (d, J ) 9.3, 1H), 8.73 (d, J ) 3.9, 1H), 8.28 (d, J
) 6.9, 1H), 7.95-7.90 (m, 1H), 7.74 (d, J ) 3.0, 1H), 7.54-
7.50 (m, 1H), 7.31-7.27 (m, 1H), 4.11 (q, J ) 6.9, 2H), 1.46 (t,
J ) 6.9, 3H). Anal. (C14H13N3O4) C, H, N.
Compound 2 and 6 were obtained from Chembridge (San
Diego, CA):
5-Br om o-N-(4-m eth oxy-2-n itr op h en yl)p yr id in e-3-ca r -
boxa m id e (2). H NMR (CDCl3): 11.12 (s, 1H), 9.11 (d, J )
1.8, 1H), 8.89 (d, J ) 2.4, 1H), 8.14 (d, J ) 9.6, 1H), 8.42 (t, J
) 2.1, 1H), 7.76 (d, J ) 3.0, 1H), 7.33 (dd, J ) 3.0, 9.3, 1H),
3.90 (s, 3H).
N-(4-Eth oxy-2-n itr oph en yl)pyr idin e-3-car boxam ide (6).
1H NMR (CDCl3): 11.12 (s, 1H), 9.24 (d, J ) 1.8, 1H), 8.85 (d,
J ) 9.3, 1H), 8.83 (dd, J ) 1.2, 5.1, 1H), 8.29-8.24 (m, 1H),
7.74 (d, J ) 3.0, 1H), 7.51-7.47 (m, 1H), 7.31 (dd, J ) 3.0,
9.3, 1H), 4.11 (q, J ) 6.9, 2H), 1.47 (t, J ) 6.9, 3H).
2-Ch lor o-N-(4-m eth oxy-2-n itr op h en yl)p yr id in e-3-ca r -
boxa m id e (3). A mixture of 2-chloronicotinoyl chloride (200
mg, 1.14 mmol), 4-methoxy-2-nitroaniline (191 mg, 1.14 mmol),
and triethylamine (160 µL) in THF (10 mL) was refluxed for
20 h. The mixture was cooled to room temperature, and the
solid was filtered. The filtrate was rotary evaporated to
dryness, and the resulting residue was purified by flash
column chromatography (40% ethyl acetate in hexanes) to
1
1
obtain a yellow solid (192 mg, 55%). H NMR (CDCl3): 10.76
(s, 1H), 8.78 (d, J ) 9.3, 1H), 8.58-8.55 (m, 1H), 8.13-8.09
(m, 1H), 7.73 (d, J ) 3.0, 1H), 7.45-7.40 (m, 1H), 7.34-7.28
(m, 1H), 3.90 (s, 3H). Anal. (C13H10ClN3O4) C, H, N.
The following compounds were prepared by a procedure
similar to that described for the preparation of compound 3.
N -(4-Me t h oxy-2-n it r op h e n yl)p yr id in e -3-ca r b oxa m -
id e (1). The title compound was prepared from nicotinoyl
chloride and 4-methoxy-2-nitroaniline. 1H NMR (CDCl3): 11.13
(s, 1H), 9.24 (d, J ) 2.1, 1H), 8.86 (d, J ) 9.3, 1H), 8.84 (dd, J
) 1.5, 5.1, 1H), 8.29-8.25 (m, 1H), 7.76 (d, J ) 3.0, 1H), 7.49
(dd, J ) 4.8, 8.1, 1H), 7.33 (dd, J ) 3.0, 9.3, 1H), 3.90 (s, 3H).
Anal. (C13H11N3O4) C, H, N.
Ca sp a se Activa tion Assa y (EC50). Human breast cancer
cell lines T47D, ZR75-1, and the human colon cancer cell line
(DLD-1) were grown according to media component mixtures
designated by American Type Culture Collection in RPMI-1640
+ 10% FCS in a 5% CO2-95% humidity incubator at 37 °C.
Cells were harvested using trypsin and washed at 600g and
resuspended at 0.65 × 106 cells/mL into RPMI media + 10%
FCS. An aliquot of 22.5 µL of cells was added to a well of a
384-well microtiter plate containing 2.5 µL of 0.05 to 100 µM
of test compound in RPMI-1640 containing 25 mM HEPES
media solution with 10% DMSO (0.005 to 10 µM final). An
aliquot of 22.5 µL of cells was added to a well of a 384-well
microtiter plate containing 2.5 µL of RPMI-1640 media solu-
tion with 10% DMSO and without test compound as the control
sample. The samples were then incubated at 37°C for 24 h in
a 5% CO2-95% humidity incubator. After incubation, the
samples were removed from the incubator and 25 µL of a
solution containing 14 µM of N-(Ac-DEVD)-N′-ethoxycarbonyl-
R110 fluorogenic substrate, 20% sucrose, 20 mM DTT, 200 mM
NaCl, 40 mM Na PIPES buffer pH 7.2, and 500 µg/mL
lysolecithin was added. The samples were incubated at room
temperature. Using a fluorescent plate reader (Model Spec-
trafour Plus Tecan), an initial reading (T ) 0) was made
approximately 1-2 min after addition of the substrate solution,
employing excitation at 485 nm and emission at 525 nm, to
determine the background fluorescence of the control sample.
After the 3 h incubation, the samples were read for fluores-
cence as above (T ) 3 h).
6-Ch lor o-N-(4-m eth oxy-2-n itr op h en yl)p yr id in e-3-ca r -
boxa m id e (4). The title compound was prepared from 6-chlo-
1
ronicotinoyl chloride and 4-methoxy-2-nitroaniline (54%). H
NMR (CDCl3): 11.13 (s, 1H), 9.01 (d, J ) 2.7, 1H), 8.83 (d, J
) 9.3, 1H), 8.24-8.20 (m, 1H), 7.76 (d, J ) 2.7, 1H), 7.52 (d,
J ) 8.7, 1H), 7.35-7.31 (m, 1H), 3.90 (s, 3H). Anal. (C13H10
ClN3O4) C, H, N.
-
N -(4-Me t h oxy-2-n it r op h e n yl)p yr id in e -4-ca r b oxa m -
id e (5). The title compound was prepared from isonicotinoyl
chloride and 4-methoxy-2-nitroaniline (40%). 1H NMR
(CDCl3): 10.79 (s, 1H), 8.83-8.81 (m, 2H), 7.85-7.83 (m, 2H),
7.61-7.55 (m, 2H), 7.40-7.36 (m, 1H), 3.87 (s, 3H). Anal.
(C13H11N3O4) H, N. C, calcd: 57.14; found: 57.93.
6-Ch lor o-N-(4-et h oxy-2-n it r op h en yl)p yr id in e-3-ca r -
boxa m id e (8). The title compound was prepared from 6-chlo-
ronicotinoyl chloride and 4-ethoxy-2-nitroaniline (34%). 1H
NMR (CDCl3): 11.13 (s, 1H), 9.01 (d, J ) 2.4, 1H), 8.82 (d, J
) 9.3, 1H), 8.24-8.20 (m, 1H), 7.74 (d, J ) 3.0, 1H), 7.51 (d,
J ) 7.8, 1H), 7.34-7.30 (m, 1H), 4.11 (q, J ) 6.9, 2H), 1.47 (t,
J ) 6.9, 3H). Anal. (C14H12ClN3O4) C, H, N.
4-Ch lor o-N-(4-eth oxy-2-n itr oph en yl)ben zam ide (9). The
title compound was prepared from 4-chlorobenzoyl chloride
and 4-ethoxy-2-nitroaniline (57%). 1H NMR (CDCl3): 11.10
(brs, 1H), 8.86 (d, J ) 9.3, 1H), 7.93 (d, J ) 8.4, 2H), 7.73 (d,
J ) 3.0, 1H), 7.51 (d, J ) 8.4, 2H), 7.32-7.27 (m, 1H), 4.11 (q,
J ) 6.9, 2H), 1.46 (t, J ) 6.9, 3H). Anal. (C15H13ClN2O4) C, H,
N.
6-Met h yl-N-(4-et h oxy-2-n it r op h en yl)p yr id in e-3-ca r -
boxa m id e (10). A mixture of 6-methylnicotinic acid (1.01 g,
7.29 mmol) and cyanuric chloride (1.34 g, 7.29 mmol) in THF
(25 mL) was stirred at room temperature for 30 min, then a
solution of 4-ethoxy-2-nitroaniline (1.33 g, 7.29 mmol) in THF
(25 mL) and triethylamine (2 mL) was added. The mixture
was refluxed for 20 h, cooled to room temperature, and then
diluted with 1:1 of hexane:ethyl acetate (200 mL). The solution
was washed with water (2 × 100 mL) and brine (100 mL), dried
over Na2SO4, and evaporated. The residue was purified by
flash column chromatography (33% ethyl acetate in hexanes)
to obtain a yellow solid (707 mg, 32%). 1H NMR (CDCl3): 11.09
(s, 1H), 9.12 (d, J ) 2.1, 1H), 8.84 (d, J ) 9.6, 1H), 8.14 (dd, J
) 2.7, 8.1, 1H), 7.73 (d, J ) 3.3, 1H), 7.34-7.28 (m, 2H), 4.11
(q, J ) 6.9, 2H), 2.67 (s, 3H), 1.46 (t, J ) 6.9, 3H). Anal.
(C15H15N3O4) C, H, N.
Ca lcu la tion . The relative fluorescence unit values (RFU)
were used to calculate the sample readings as follows:
RFU(T)3h) - control RFU(T)0) ) net RFU(T)3h)
The activity of caspase activation was determined by the
ratio of the net RFU value for the test compound to that of
control samples. The EC50 (µM) was determined by a sigmoidal
dose-response calculation (XLFit3, IDBS), as the concentra-
tion of compound that produces the 50% maximum response.
The caspase activation activity (EC50) in three cancer cell lines,
T47D, ZR75-1, and DLD-1, are summarized in Table 1.
Mor p h ologica l Assessm en t of Nu clea r F r a gm en ta tion
of Ap op totic Cells. J urkat cells, grown and harvested as
above, were treated with 5 µM of compound 1 for 24 h followed
by staining of the nucleus with Syto16, a fluorescent DNA dye.
The cells were then observed under a fluorescent microscope
for chromosomal condensation and nuclear fragmentation (490
nm). The nuclei of J urkat cells treated with vehicle control
(DMSO) are seen to be round with dispersed chromatin that
is moderately stained with Syto16 (Figure 1A). In contrast,
J urkat cells treated with 5 µM of compound 1 have shrunken
and fragmented nuclei (Figure 1B), which is a hallmark of
caspase-mediated apoptosis, as well as condensed mitotic
spindles. These results corroborate the caspase activation
assays, showing that compound 1 can induce a key cellular
marker of apoptosis.
N-(4-Eth oxy-2-n itr oph en yl)pyr idin e-2-car boxam ide (7).
The title compound was prepared from picolinic acid and
4-ethoxy-2-nitroaniline by a procedure similar to that described
Cell Cycle An a lysis a n d Mea su r em en t of Ap op tosis.
T47D cells were maintained and harvested as described above.

2480 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 12
Cai et al.
Cells (1 × 106) were treated with 10 µM of compound 1 for 48
h at 37 °C. As a control, cells were also incubated with an
equivalent amount of solvent (DMSO). Cells were harvested
at 1200 rpm and washed twice with 5 mM EDTA/PBS. Cells
were then resuspended in 300 µL EDTA/PBS and 700 µL of
100% ethanol, vortexed, and incubated at room temperature
for 1 h. Samples were centrifuged at 1200 rpm for 5 min, and
the supernatant was removed. A solution containing 100 µg/
mL of propidium iodide and 1 mg/mL of RNAse A (fresh) was
added to the samples and incubated for 1 h at room temper-
ature. Samples were then transferred to 12 × 75 mm poly-
styrene tubes and analyzed on a flow cytometer. All flow
cytometry analyses were performed on a FACScalibur (Becton
Dickinson) using Cell Quest analysis software. On the x-axis
is plotted the fluorescence intensity and on the y-axis is plotted
the number of cells with that fluorescence intensity. The T47D
control cell population profile is shown in Figure 2A with most
of the cells in the M2 phase. An increase in the G2/M DNA
content (M4) of cells from 34.8% to 57.2% was observed when
cells were treated with compound 1 at 10 µM for 48 h (Figure
2B). Concurrently, an increase in the sub-diploid DNA content
of cells (marker M1 region, Figure 2B) from 2.6% to 25.1% was
observed with compound treatment. The sub-diploid amount
of DNA (M1) is indicative of apoptotic cells which have
undergone DNA degradation and nuclear fragmentation.
In a separate experiment, T47D cells were treated with 1
µM of compound 8 and measured in a similar manner as that
of compound 1. Cells treated with compound 8 at 1 µM for 48
h (Figure 3B) were accumulated in the G2/M phase (M4), from
16.5% to 18.5%. In addition, the sub-G1 population of cells with
reduced DNA content (M1) was increased substantially over
control cells (Figure 3A), from 3.3% to 56.1%. These data
indicate that treatment of T47D cells with compound 1, and
its more potent analogue 8, results in cell cycle arrest in the
G2/M phase and induction of apoptosis.
Cell Gr ow th In h ibition Assa ys (GI50). Cells were grown
and harvested as described above. An aliquot of 45 µL of cells
(4.4 × 104 cells/mL) were added to a well of a 96-well microtiter
plate, then 5 µL of 0.01 to 100 µM of test compound (0.001 to
10 µM final concentration) in RPMI-1640 media solution with
10% DMSO was added. An aliquot of 45 µL of cells were added
to a well of a 96-well microtiter plate containing 5 µL of RPMI-
1640 media solution with 10% DMSO and without test
compound as the control sample for maximal cell proliferation
(Amax). The samples were then incubated at 37 °C for 48 h in
a 5% CO2-95% humidity incubator. After incubation, the
samples were removed from the incubator and 25 µL of
CellTiter-Glo reagent (Promega) was added. The samples were
mixed by agitation and incubated at room temperature for 10-
15 min. Plates were then read using a luminescent plate reader
(Model Spectrafluor Plus Tecan Instrument) to give Atest value.
each experimental compound (from a 100× stock) in a 96-well
was added 99 µL of supplemented tubulin supernatant.
Incubation was done in a Molecular Devices plate reader at
37 °C, and absorbance readings at 340 nm were recorded every
minute for an hour. The IC50 for tubulin inhibition was the
concentration found to decrease the initial rate of tubulin
polyeriztion by 50% as calculated with Prism 2.0.
Ack n ow led gm en t. The authors acknowledge the
fine technical support given by Regina Brand and Ryan
Yoshimura.
Refer en ces
(1) Reed, J . C.; Tomaselli, K. J . Drug Discovery Opportunities From
Apoptosis Research. Curr. Opin. Biotechnol. 2000, 11, 586-592.
(2) Reed, J . C. Dysregulation of Apoptosis in Cancer. J . Clin. Oncol.
1999, 17, 2941-2953.
(3) Robertson, G. S.; Crocker, S. J .; Nicholson, D. W.; Schulz, J . B.
Neuroprotection by the Inhibition of Apoptosis. Brain Pathol.
2000, 10, 283-292.
(4) Leung, D.; Abbenante, G.; Fairlie, D. P. Protease Inhibitors:
Current Status and Future Prospects. J . Med. Chem. 2000, 43,
305-41.
(5) Thornberry, N. A. Caspases: Key Mediators of Apoptosis. Chem.
Biol. 1998, 5, R97-R103.
(6) Kolenko, V. M.; Uzzo, R. G.; Bukowski, R.; Finke, J . H. Caspase-
Dependent and -Independent Death Pathways in Cancer
Therapy. Apoptosis 2000, 5, 17-20.
(7) (a) Simoni, D.; Invidiata, F. P.; Rondanin, R.; Grimaudo, S.;
Cannizzo, G.; Barbusca, E.; Porretoo, F.; D’Alessandro, N.;
Tolomeo, M. Structure-Activity Relationship Studies of Novel
Heteroretinoids: Induction of Apoptosis in the GL-60 Cell Line
by a Novel Isoxazole-Containing Heteroretinoid. J . Med. Chem.
1999, 42, 4961-4969. (b) Montero, E. I.; Diaz, S.; Gonzalez-
Vadillo, A. M.; Perez, J . M.; Alonso, C.; Navarro-Ranninger, C.
Preparation and Characterization of Novel trans-[PtCl2(amine)-
(isopropylamine)] Compounds. Cytotoxic Activity and Apoptosis
Induction in ras-Transformed Cells. J . Med. Chem. 1999, 42,
4264-4268. (c) Simoni, D.; Roberti, M.; Invidiata, F. P.; Ron-
danin, R.; Baruchello, R.; Malagutti, C.; Mazzali, A.; Rossi, M.;
Grimaudo, S.; Capone, F.; Dusonchet, L.; Meli, M.; Raimondi,
M. V.; Landino, M.; D′Alessandro, N.; Tolomeo, M.; Arindam,
D.; Lu, S.; Benbrook, D. M. Heterocycle-Containing Retinoids.
Discovery of a Novel Isoxazole Arotinoid Possessing Potent
Apoptotic Activity in Multidrug and Drug-Induced Apoptosis-
Resistant Cells. J . Med. Chem. 2001, 44, 2308-2318. (d)
J akobsen, C. M.; Denmeade, S. R.; Isaacs, J . T.; Gady, A.; Olsen,
C. E.; Christensen, S. B. Design, Synthesis, and Pharmacological
Evaluation of Thapsigargin Analogues for Targeting Apoptosis
to Prostatic Cancer Cells. J . Med. Chem. 2001, 44, 4696-4703.
(e) Gonzales, A. G.; Silva, M. H.; Padron, J . I.; Leon, F.; Reyes,
E.; Alvarez-Mon, M.; Pivel, J . P.; Quintana, J .; Estevez, F.;
Bermejo, J . Synthesis and Antiproliferative Activity of a New
Compound Containing an R-Methylene-γ-Lactone Group. J . Med.
Chem. 2002, 45, 2358-2361.
(8) Weber, E.; Tseng, B. Y.; Drewe, J . A.; Cai, S. X. Methods of
Identifying Potentially Therapeutically Effective Antineoplastic
Agents with Viable Cultured Cells Having an Intact Cell
Membrane and Product by Same. Patent Application No.
WO0045165, 2000.
(9) Cai, S. X.; Zhang, H.-Z.; Guastella, J .; Drewe, J .; Yang, W.;
Weber, E. Design and Synthesis of Rhodamine 110 Derivative
and Caspase-3 Substrate for Enzyme and Cell-Based Fluorescent
Assay. Bioorg. Med. Chem. Lett. 2001, 11, 39-42.
(10) For the preparation of the substrate, please refer to: Cai, S. X.;
Keana, J . F. W.; Drewe, J . A.; Zhang, H.-Z. Fluorogenic or
Fluorescent Reporter Molecules and Their Applications for
Whole-Cell Fluorescence Screening Assays for Caspases and
Other Enzymes and the Use Thereof. US Patent No. 6, 335, 429,
column 116, example 25, column 117, example 30 and 31, 2002.
(11) McGovern, S. L.; Caselli, E.; Grigorieff, N.; Shoichet, B. K. A
Common Mechanism Underlying Promiscuous Inhibitors From
Virtual and High-Throughput Screening. J . Med. Chem. 2002,
45, 1712-1722.
Baseline for GI50 (dose for 50% inhibition of cell prolifera-
tion) of initial cell numbers was determined by adding an
aliquot of 45 µL of cells and 5 µL of RPMI-1640 media solution
with 10% DMSO to wells of a 96-well microtiter plate. The
samples were then incubated at 37 °C for 0.5 h in a 5% CO2-
95% humidity incubator. After incubation, the samples were
removed from the incubator and 25 µL of CellTiter-Glo reagent
(Promega) was added. The samples were mixed by agitation
and incubated at room temperature for 10-15 min. Lumines-
cence was read as above to give Astart value, defining lumines-
cence for initial cell number used as baseline in GI50 deter-
minations.
Ca lcu la tion . GI50 (dose for 50% inhibition of cell prolifera-
tion) is the concentration where [(Atest - Astart)/(Amax - Astart)]
) 0.5. The GI50 (µM) of compound 1, 6, and 10 in T47D and
DLD-1 cells are summarized in Table 2 in comparison with
the caspase activition activity (EC50). The GI50 (µM) of com-
pound 10 in MES-SA and MES-SA/DX5 cells are summarized
in Table 3 in comparison with that of paclitaxel.
(12) Wood, K. W.; Cornwell, W. D.; J ackson, J . R. Past and Future
of the Mitotic Spindle as An Oncology Target. Curr. Opin.
Pharmacol. 2001, 1, 370-377.
(13) Rubenstein, S. M.; Baichwal, V.; Beckmann, H.; Clark, D. L.;
Frankmoelle, W.; Roche, D.; Santha, E.; Schwender, S.; Thoolen,
M.; Ye, Q.; J aen, J . C. Hydrophilic, Pro-Drug Analogues of
T138067 are Efficacious in Controlling Tumor Growth In Vivo
and Show a Decreased Ability to Cross the Blood Brain Barrier.
J . Med. Chem. 2001, 44, 3599-3605.
Tu bu lin P olym er iza tion Assa y. Lyophilized tubulin (Cy-
toskeleton #ML113, 1 mg, MAP-rich) was assayed for the effect
of the test compound on tubulin polyermization according to
the recommended procedure of the manufacturer. To 1 µL of

N-Phenyl Nicotinamides as Inducers of Apoptosis
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 12 2481
(14) J ordan, M. A. Mechanism of Action of Antitumor Drugs that
Interact with Microtubules and Tubulin. Curr. Med. Chem.
Anticancer Agents 2002, 2, 1-17.
(17) (a) Medina, J . C.; Roche, D.; Shan, B.; Leaner, R. M.; Frank-
moelle, W. P.; Clark, D. L.; Rosen, T.; J aen, J . C. Novel
Halogenated Sulfonamides Inhibit the Growth of Multidrug
Resistant MCF-7/ADR Cancer Cells. Bioorg. Med. Chem. Lett.
1999, 9, 1843-1846. (b) Medina, J . C.; Shan, B.; Beckman, H.;
Farrell, R. P.; Clark, D. L.; Learned, M.; Roche, D.; Li, A.;
Baichwal, V.; Case, C.; Baeuerle, P. A.; Rosen, T.; J aen, J . C.
Novel Antineoplastic Agents with Efficacy Against Multidrug
Resistant Tumor Cells. Bioorg. Med. Chem. Lett. 1998, 8,
2653-2656.
(15) Kaye, S. B. Multidrug Resistance: Clinical Relevance in Solid
Tumors and Strategies For Circumvention. Curr. Opin. Oncol.
1998, 10, S15-19.
(16) Shan, B.; Medina, J . C.; Santha, E.; Frankmoelle, W. P.; Chou,
T. C.; Learned, R. M.; Narbut, M. R.; Stott, D.; Wu, P.; J aen, J .
C.; Rosen, T.; Timmermans, P. B.; Beckmann, H. Selective
Covalent Modification of Beta-Tubulin Residue Cys-239 by
T138067, an Antitumor Agent with In Vivo Efficacy Against
Multidrug-Resistant Tumors. Proc. Natl. Acad. Sci. U.S.A. 1999,
96, 5686-5691.
J M0205200